The handle
http://hdl.handle.net/1887/136525
holds various files of this Leiden University
dissertation.
Author: Boeters, D.M.
Resolution of rheumatoid
arthritis
Does immunological remission,
defined as disappearance of
autoantibodies, occur with
current treatment strategies?
A long-term follow-up study in
rheumatoid arthritis patients
who achieved sustained
DMARD-free status
Ann Rheum Dis. 2019;78(11):1497-1504.
Debbie M. Boeters Leonie E. Burgers René E.M. Toes
Annette van der Helm-van Mil
Abstract
Objectives
Sustained disease-modifying antirheumatic drug (DMARD)-free status, the sustained absence of synovitis after cessation of DMARD therapy, is infrequent in autoantibody-positive rheumatoid arthritis (RA), but approximates cure (i.e., disappearance of signs and symptoms). It was recently suggested that immunological remission, defined as disappearance of anti-citrullinated protein antibodies (ACPA) and rheumatoid factor (RF), underlies this outcome. Therefore, this long-term observational study determined if autoantibodies disappear in RA patients who achieved sustained DMARD-free remission.
Methods
We studied 95 ACPA-positive and/or RF-positive RA patients who achieved DMARD-free remission after median 4.8 years and kept this status for the remaining follow-up (median 4.2 years). Additionally, 21 autoantibody-positive RA patients with a late flare, defined as recurrence of clinical synovitis after a DMARD-free status of ≥1 year, and 45 autoantibody-positive RA patients who were unable to stop DMARD therapy (during median 10 years) were studied. Anti-cyclic citrullinated peptide 2 (anti-CCP2) IgG, IgM and RF IgM levels were measured in 587 samples obtained at diagnosis, before and after achieving DMARD-free remission.
Results
13% of anti-CCP2 IgG-positive RA patients had seroreverted when achieving remission. In RA patients with a flare and persistent disease this was 8% and 6%, respectively (p=0.63). For anti-CCP2 IgM and RF IgM, similar results were observed. Evaluating the estimated slope of serially measured levels revealed that RF levels decreased more in patients with than without remission (p<0.001); the course of anti-CCP2 levels was not different (p=0.66).
Conclusions
Sustained DMARD-free status in autoantibody-positive RA was not paralleled by an increased frequency of reversion to autoantibody negativity. This form of immunological remission may therefore not be a treatment target in patients with classified RA.
Introduction
Sustained disease-modifying antirheumatic drug (DMARD)-free status is defined as sustained absence of synovitis after cessation of all DMARD therapy and is increasingly achievable by patients with rheumatoid arthritis (RA).1 This status
is also characterised by normalisation of functional status and lower levels of fatigue, pain and morning stiffness and is currently considered the best possible outcome of RA.1 Absence of anti-citrullinated protein antibodies (ACPA) at disease
presentation is an important predictor of achievement of sustained DMARD-free remission; however, with current treatment strategies this outcome is also observed in 10% of ACPA-positive RA.1-5
The pathophysiological role of ACPA in RA development or progression is not exactly known. ACPA can be present years before the onset of joint symptoms and disease, indicating that the mere presence of ACPA is not enough to develop disease.6,7 Studies in the preclinical phase have shown that the ACPA immune
response matures once disease onset is approached, as characterised by an increase in ACPA level, isotype-usage, avidity and the number of citrullinated epitopes recognised by ACPA.8-11 In addition, there are changes in Fc glycosylation before
RA onset.12 Once RA is established the ACPA immune response does not mature any
further.13 Besides ACPA, also rheumatoid factor (RF) can be present years before
disease onset.6,7 Since autoantibodies are considered to have a prominent role in
seropositive RA and precede symptom development, it is tempting to hypothesise that changes in the autoantibody response occur before or at the time when clinical disease has been extinguished, as is the case when sustained DMARD-free remission is reached. In this light, it was recently suggested that disappearance of autoantibodies is a hallmark of immunological remission and might characterise patients who are able to achieve drug-free remission.14
However, so far this hypothesis has not been thoroughly investigated. In a few studies, seroconversion and seroreversion during follow-up of early arthritis and RA patients were investigated. The observations described indicate that both are infrequent and not associated with relevant outcomes such as radiographic damage, functional status or the disease activity score.15-18 In only one study, the
association between seroreversion and drug-free remission was analysed and no association was observed.19 However, autoantibody levels were only determined
at disease presentation and at 1 year of follow-up, thus generally years before achievement of drug-free remission. In addition, follow-up of patients after the
achievement of drug-free remission was limited.19
We aimed to increase the understanding of the long-term course of RA-related autoantibodies in patients who had achieved the closest available proxy of cure of RA. Therefore, we investigated the association between ACPA and RF seroreversion and achievement of sustained DMARD-free remission in a unique population of RA patients with available serum samples at the time of remission and with a long follow-up duration after achievement of DMARD-free status.
Methods
Patients
Patients were retrieved from the Leiden Early Arthritis Clinic cohort, which is an inception cohort that includes patients with clinically confirmed arthritis and symptom duration <2 years. At baseline, patients and rheumatologists completed questionnaires, joint counts were performed and blood samples were collected. Follow-up visits were scheduled and blood samples were taken at 3-4 months, 6-8 months, 12 months, 18 months, 24 months and yearly thereafter. Between 1993 and 2014, 3473 patients were consecutively included, of which 1586 patients had a clinical diagnosis of RA and also fulfilled 1987 or 2010 RA classification criteria during the first year of follow-up.20,21 Of these, 941 patients were ACPA-positive
and/or RF-positive at baseline.
Treatment strategies changed over time. In general, patients included in 1993-1995 were initially treated with non-steroidal anti-inflammatory drugs, patients included in 1996-1998 with mild DMARDs (hydroxychloroquine or sulphasalazine) and patients included ≥1999 were initially treated with methotrexate. When this treatment failed, another conventional DMARD was initiated or added. A biological DMARD was allowed in patients who failed on ≥2 conventional DMARDs. Medication used by all studied patients during the observed follow-up period is shown in Table 9.1. Disease activity score (DAS44)-guided treatment became common from 2005 onwards with tapering and eventually stopping of treatment if DAS44 remained <2.4 and synovitis was absent at clinical joint examination, and intensifying treatment in case of DAS44 ≥2.4.
Table 9.1 Medication use during the total follow-up period; stratified by patient group
DMARD-free sustained
remission (n=95) Flare after DMARD-free remission (n=21) Persistent RA(n=45)
Methotrexate, n(%) 76 (80) 21 (100) 44 (98)
Other conventional DMARDs, n(%) 46 (48) 11 (52) 37 (82)
Sulfasalazine, n(%) 27 (28) 6 (29) 28 (62) Hydroxychloroquine, n(%) 26 (27) 8 (38) 32 (71) Leflunomide, n(%) 9 (9) 5 (24) 17 (38) Azathioprine, n(%) 1 (1) 0 (0) 3 (7) Cyclosporine, n(%) 0 (0) 1 (5) 0 (0) Gold, n(%) 2 (2) 0 (0) 1 (2) Biological DMARD, n(%) 14 (15) 6 (29) 19 (42) TNF-inhibitor, n(%) 11 (12) 4 (19) 17 (38) Rituximab, n(%) 1 (1) 1 (5) 2 (4) Abatacept, n(%) 0 (0) 0 (0) 3 (7) Tocilizumab, n(%) 2 (2) 1 (5) 3 (7) Omalizumab, n(%) 0 (0) 0 (0) 1 (2)
Numbers indicate the number of patients who used the indicated medication at any time during follow-up; therefore, the indicated percentages for the different groups do not add up to 100%. The duration that patients used the medication and the number of patients using combination therapy is not indicated here. DMARD, disease-modifying antirheumatic drug; RA, rheumatoid arthritis; TNF, tumor necrosis factor.
Of the 1586 RA patients, medical files were studied on occurrence of sustained DMARD-free remission until April 2017. This outcome was defined as absence of synovitis (by physical examination) after cessation of all DMARD therapy (including biologics and systemic and intra-articular corticosteroids) for at least 1 year and for the remainder of follow-up. Patients who experienced a flare of clinical synovitis early or late after DMARD cessation were considered as not in sustained DMARD-free remission. The date of sustained DMARD-DMARD-free remission was the date 1 year after DMARD cessation. Patients who did not achieve remission were censored at the date when medical files were explored or at an earlier date when they were lost to follow-up or had died. Ninety-five of 941 ACPA-positive and/or RF-positive RA patients achieved sustained DMARD-free remission after a median follow-up of 4.8 years (Figure 9.1). After achievement of sustained DMARD-free remission, patients were additionally followed for median 4.2 years. Except for 1 patient, all patients were included from 1999 onwards.
Figure 9.1 Flowchart of patient selection
DMARD-free remission was defined as the absence of clinical synovitis for ≥1 year after DMARD cessation. Flares were defined as recurrence of synovitis after having achieved DMARD-free remission, thus recurrence of synovitis >1 year after DMARD cessation. Sustained DMARD-free remission was defined as absence of synovitis after DMARD cessation during the complete follow-up, but at least for 1 year. As control, 45 autoantibody-positive patients with persistent RA were selected from the group of autoantibody-positive RA patients who never achieved DMARD-free remission based on comparable inclusion period as patients who achieved sustained remission and based on available serum samples. ACPA, anti-citrullinated protein antibodies; DMARD, disease-modifying antirheumatic drug; RA, rheumatoid arthritis; RF, rheumatoid factor.
who never achieved DMARD-free remission based on comparable inclusion period and on available serum samples at baseline, 1 year and at 7-8 years follow-up or earlier in case patients had a shorter follow-up duration.
Median total follow-up of the studied patients was 10 years and was comparable between the studied groups.
Serological measurements
Anti-cyclic citrullinated peptide 2 (anti-CCP2) IgG and RF IgM were measured in 587 serum samples obtained at diagnosis, before and after achieving DMARD-free remission, using enzyme-linked immunosorbent assays as described previously.22,23 In all cases, <5% of controls were autoantibody-positive with the
cut-offs used. In short, for IgM-RF, human IgG1 was used as the capture antigen and bound antibodies were detected with F(ab′)2 fragments of peroxidase-conjugated antihuman IgM. The cut-off for positivity was 8 IU/mL. For anti-CCP2 IgG, the anti-CCP2 test (Immunoscan RA Mark 2; Euro-Diagnostica, Arnhem, The Netherlands) was used with a cut-off value of 25 units/mL, as described in the manufacturer’s instructions. An overview of the measured samples during follow-up for the different grofollow-ups is depicted in Figure 9.2. Of each patient, a median of three samples was measured. In addition, anti-CCP2 IgM was measured in the first and last available serum sample of patients who were positive for anti-CCP2 IgG at disease presentation. In brief, microtiter plates were coated with citrullinated CCP2. An arginine control was used as control for citrulline specificity of the anti-CCP antibodies. Plates were incubated for 1 hour at 37°C with serum samples, 50 µL/well, at a dilution of 1:50. To detect anti-CCP2 IgM, plates were incubated for 1 hour at 37°C, 50 µL/well, with peroxidase-conjugated anti-human IgM. Pooled serum samples of highly positive patients were used in all plates to generate standard curves. Autoantibody levels were estimated by interpolation from these standard curves and were expressed in arbitrary units per millilitre. Samples were considered positive when the signal was higher than the mean +2 SD of serum samples of 64 healthy control subjects in total. This resulted in a cut-off value of 12 AU/mL. In addition, to ascertain that the obtained signal within the anti-CCP2 IgM ELISA was citrulline-specific, the difference between the signal against the citrullinated peptide and the unmodified arginine peptide had to be >0.1 (OD >0.1).
Figure 9.2 Overview of samples measured during follow-up of patients who achieved sustained DMARD-free remission (A), patients with a late flare (B) and patients with persistent RA (C) Numbers indicate the number of samples measured within the indicated time periods. In total, 587 samples were measured. Of patients who achieved sustained DMARD-free remission, samples were obtained at diagnosis, before and at or after achieving remission. DMARD, disease-modifying antirheumatic drug; RA, rheumatoid arthritis.
Statistical analyses
The difference between the first and last available serum sample was used to calculate the proportion of patients with seroreversion. This was performed separately for patients positive for anti-CCP2 IgG, anti-CCP2 IgM and RF IgM. Differences in seroreversion between the three patient groups were compared with the Fisher exact test. To test whether changes in ACPA and/or RF levels during follow-up were associated with achievement of sustained DMARD-free remission, Cox proportional hazards regression analyses were performed with time till achievement of remission as outcome. For these analyses, patients with a late flare were combined with patients with persistent RA as one group. Changes in anti-CCP2 IgG and RF IgM level per year were estimated with linear regression analyses for each patient individually. These changes in levels over time were used as predictor in Cox proportional hazards regression analyses. Antibody levels below the detection limit were imputed with a value of 0.
Several subanalyses were performed to study whether results on seroreverion could be ascribed to variation around the cut-off level, whether results were different when groups were stratified for autoantibody combinations, whether results were different in patients who were treated early, or whether results were dependent on the follow-up duration. Finally, for RF a second cut-off for RF positivity was used. This was done as the cut-off that is used in clinical practice has a specificity of 95% when compared with healthy controls, but a reduced specificity when patients with other arthritides were used as reference.24 A cut-off
of 33 allowed a specificity of 98% relative to patients with other early arthritides in our cohort (data not shown) which was then equal to the specificity of ACPA. SPSS V.23.0 was used and p-values <0.05 were considered significant.
Results
Patient characteristics
Baseline characteristics of the studied autoantibody-positive patients are presented in Table 9.2 and are similar between the different groups, with the exception that patients who achieved sustained DMARD-free remission were less frequently ACPA-positive.
Table 9.2 Baseline characteristics of RA patients who achieved sustained DMARD-free remission, who flared after being in DMARD-free remission and of patients with persistent RA
Sustained DMARD-free remission (n=95)
Late flare after ≥1 year of DMARD-free
remission (n=21)
Persistent RA (n=45)
p-value
Age in years, mean (SD) 54 (17) 52 (13) 55 (12) 0.63
Female, n (%) 65 (68) 13 (62) 30 (67) 0.85
Symptom duration in weeks, median (IQR)
17 (10-35) 20 (8-37) 20 (13-38) 0.56 66-SJC, median (IQR) 5 (3-9) 5 (2-13) 5 (3-8) 0.81 68-TJC, median (IQR) 8 (3-13) 12 (4-15) 5 (4-11) 0.32
Autoantibody status 0.047
anti-CCP2 IgG+ RF IgM-, n(%) 9 (9) 0 (0) 4 (9) anti-CCP2 IgG- RF IgM+, n(%) 41 (43) 6 (29) 10 (22) anti-CCP2 IgG+ RF IgM+, n(%) 45 (47) 15 (71) 31 (69)
CRP (mg/L), median (IQR) 11 (3-28) 11 (4-23) 14 (5-40) 0.39 ESR (mm/h), median (IQR) 26 (14-49) 29 (13-44) 29 (20-41) 0.75
Patients with persistent RA were selected from the group of autoantibody-positive RA patients who never achieved DMARD-free remission based on comparable inclusion period as patients who achieved sustained remission and based on available serum samples. anti-CCP2, anti-cyclic citrullinated peptide 2; CRP, C-reactive protein; DMARD, disease-modifying antirheumatic drug; IQR, interquartile range; RA, rheumatoid arthritis; RF, rheumatoid factor; SD, standard deviation; SJC, 66-swollen joint count; symptom duration, time between symptom onset and inclusion in cohort; TJC, 68-tender joint count.
Anti-CCP2 IgG and RF IgM seroreversion were not associated with achievement of sustained DMARD-free remission
First, anti-CCP2 IgG levels were serially measured in the three different patient groups (Figure 9.3A). Of anti-CCP2 IgG-positive RA patients who achieved sustained DMARD-free remission, 13% had reverted to anti-CCP2 IgG negativity around the time of remission (Figure 9.4A). However, for RA patients with a late flare or with persistent disease, seroreversion was observed in 8% and 6%, respectively, which was not significantly different from patients who achieved sustained DMARD-free remission (p=0.63).
Figure 9.3 Anti-CCP2 IgG levels in ACPA-positive RA patients (A) and RF IgM levels in RF-positive patients (B) during follow-up, stratified for clinical outcome
Dotted lines indicate the cut-off values (25 AU/mL for anti-CCP2 IgG and 8 IU/mL for RF IgM). Values below the detection limit were imputed with the value of 0. Number of ACPA and RF-positive patients in each group: DMARD-free sustained remission: ACPA+ n=54, RF+ n=86, flare: ACPA+ n=15, RF+ n=21, persistent RA: ACPA+ n=35, RF+ n=41. ACPA, anti-citrullinated protein antibodies; anti-CCP2, anti-cyclic citrullinated peptide 2; DMARD, disease-modifying antirheumatic drug; RA, rheumatoid arthritis; RF, rheumatoid factor.
Patients with seroreversion had lower median anti-CCP2 IgG levels at disease presentation than patients without seroreversion (42 and 420 AU/mL, respectively, p<0.001). Ever use of biological DMARDs was comparable in patients with and without seroreversion (33% and 32%, respectively, p=1.00). To further analyse the effect of differences in medication use between patients, analyses were stratified for medication ever used during follow-up. This revealed similar results as in the whole group of patients (Supplementary table 9.1).
Similar results were observed for RF IgM (Figures 9.3B and 9.4B). RF-positive patients who achieved sustained DMARD-free remission had seroreversion in 20%, whereas this occurred in 6% and 15% of patients with a late flare and with persistent disease, respectively (p=0.44, Figure 9.4B). RF IgM levels were lower in patients with seroreversion than in patients who remained positive for RF IgM (19 and 53 IU/mL, respectively, p=0.003). Thus, ACPA or RF seropositive RA patients who achieved sustained DMARD-free remission did not become more frequently seronegative than patients who did not achieve remission.
Figure 9.4 Reversion to anti-CCP2 IgG (A) and RF IgM (B, C) seronegativity in autoantibody-positive RA patients who achieved sustained DMARD-free remission, who had a late flare and in patients with persistent RA
Analyses were performed in patients positive for anti-CCP2 IgG (A) or positive for RF IgM (B, C). For the analyses presented in (A) and (B) the cut-offs used were determined by the manufacturer and are similar to those used in clinical practice. For RF, also a cut-off of 33 was used to define positivity. This cut-off resulted in a specificity of 98% relative to patients with other early arthritides and was then comparable to the ACPA test (C). Seroreversion was defined as shifting from seropositive at baseline to seronegative in the last available serum sample; for patients who achieved sustained DMARD-free remission the last sample was measured at the time of remission. Number of patients in each group: sustained DMARD-free remission: ACPA+ n=47, RF+ cut-off 8 n=71, RF+ cut-off 33 n=44, late flare: ACPA+ n=12, RF+ cut-off 8 n=16, RF+ cut-off 33 n=13, persistent RA: ACPA+ n=35, RF+ cut-off 8 n=41, RF+ cut-off 33 n=25. ACPA, anti-citrullinated protein antibodies; anti-CCP2, anti-cyclic anti-citrullinated peptide 2; DMARD, disease-modifying antirheumatic drug; RA, rheumatoid arthritis; RF, rheumatoid factor.
Changes in RF IgM levels were larger in patients with sustained DMARD-free remission than in patients with persistent RA
16% (p<0.001). Thus, seropositive RA patients who achieved sustained DMARD-free remission did not have disappearance of autoantibodies; however, there was a significant decrease of RF IgM levels in patients who achieved sustained DMARD-free remission compared with patients who did not.
Anti-CCP2 IgM seroreversion was not associated with sustained DMARD-free remission
Finally, the proportion of patients seroreverting from anti-CCP2 IgM positive to negative was studied as we hypothesised that if the ACPA immune response had changed in patients who achieved remission, this could be reflected by a decreased presence of anti-CCP2 IgM, since IgM is an indication of an ongoing immune response. Of CCP2 IgG-positive patients, 25-29% were also positive for anti-CCP2 IgM at disease presentation within the different groups. During follow-up, 31% (4/13) of the anti-CCP2 IgG and IgM-positive patients who achieved DMARD-free remission seroreverted from positive to negative anti-CCP2 IgM (Figure 9.5). For patients with a late flare and patients with persistent RA, this occurred in 100% (3/3) and 60% (6/10), respectively. Thus, patients who achieved sustained DMARD-free remission and who were seropositive for anti-CCP2 IgM at disease presentation did not serorevert more frequently than patients who did not achieve remission.
Figure 9.5 Change in anti-CCP2 IgM status during follow-up in patients who achieved DMARD-free sustained remission, who had a flare and in patients with persistent RA
Data are shown for the subgroup of anti-CCP2 IgG+ patients at baseline. Number of patients in each group: DMARD-free sustained remission: n=47, flare: n=12, persistent RA: n=35. Anti-CCP2, anti-cyclic citrullinated peptide 2; DMARD, disease-modifying antirheumatic drug; RA, rheumatoid arthritis.
Sensitivity analyses
To investigate whether our results were driven by patients with autoantibody levels fluctuating around the cut-off, analyses were performed in patients with baseline autoantibody levels above the median which showed that none of the anti-CCP2 IgG-positive patients had seroreverted. For RF IgM, 8% of patients who achieved sustained DMARD-free remission, none of the patients with a late flare and 10% of patients with persistent disease had seroreverted.
To study the hypothesis that early treatment might be associated with higher rates of seroreversion, analyses were repeated in patients with short symptom duration before treatment start (<12 weeks) and with long symptom duration (≥12 weeks). Of anti-CCP2 IgG-positive patients who achieved remission, 10% of the patients with a short symptom duration had reverted to seronegativity; this was 15% of patients with a long symptom duration (p=1.00). For RF IgM, these percentages were 25% and 18%, respectively (p=0.54). Thus, seroreversion rates were not higher in patients with earlier treatment initiation.
Next, it was assessed whether single autoantibody positivity was associated with higher seroreversion rates. Of ACPA-positive patients, 7% of ACPA+ RF+ and 23% of ACPA+ RF- patients had reverted from anti-CCP2 IgG positivity to negativity (p=0.12). Of RF-positive patients, 6% of ACPA+ RF+ and 34% of ACPA- RF+ patients had reverted to RF IgM negativity (p<0.001).
In addition, analyses were stratified for different autoantibody combinations, since patients with sustained DMARD-free remission were less frequently positive for both ACPA and RF than the other studied groups. Similar to our main analysis, seroreversion rates for ACPA and RF were not significantly different between patients with and without sustained remission (Supplementary table 9.2). Thus, single RF-positive patients seroreverted more often (when cut-off of 8 was used) than ACPA+ RF+ patients, but the frequency of seroreversion was not higher in the group that achieved sustained remission group compared with the group that did not.
CCP2 IgG-positive RA patients who achieved sustained DMARD-free remission, 6% had reverted to anti-CCP2 negativity around the time of remission, which was not different from patients with a late flare and with persistent disease, who had seroreversion in 8% and 6%, respectively (p=1.00). Of RF IgM-positive RA patients who achieved sustained DMARD-free remission, 16% had reverted to RF IgM negativity. Of patients with a late flare and with persistent disease this occurred in 6% and 15%, respectively (p=0.78).
Finally, seroreversion for RF was also studied when the cut-off for positivity was set at 33. Also then no differences in seroreversion rates were observed between the three different groups (p=0.86, Figure 9.4C). When the different autoantibody combinations were studied with this cut-off, 6% of ACPA+ RF+ and 16% of ACPA+ RF- patients had reverted to anti-CCP2 negativity (p=0.27). Of RF-positive patients, 37% of ACPA+ RF+ and 35% of ACPA- RF+ patients had reverted to RF IgM negativity (p=1.00). Thus, also when a different cut-off for RF positivity was used, patients who achieved sustained DMARD-free remission did not serorevert more often than patients with a late flare or with persistent disease.
Discussion
Currently, a sustained DMARD-free status is the best possible clinical outcome as, per definition, clinically apparent synovitis is persistently absent and patients in this status also have resolution of symptoms and normalised functional status.1
This outcome is achievable in autoantibody-positive RA, although with a lower frequency than in autoantibody-negative RA. The biological nature underlying this type of persistent remission is unknown. It was recently suggested that it is characterised by disappearance of autoantibodies.14 The present large observational
study with a unique, long follow-up period and with samples measured at the time of remission, explored this hypothesis. No association between remission and reversion to autoantibody negativity was demonstrated. Hence, although it has been suggested that immunological remission is characterised by disappearance of autoantibodies,14 we studied patients in the deepest form of clinical remission
and observed no increased frequency of reversion to seronegativity in this group. To our knowledge, this is the first study in which seroreversion rates in patients with long-standing DMARD-free status were investigated. Importantly, for both ACPA and RF seroreversion was infrequent and not related to clinical outcome. In
previous studies in patients with established RA who were treated with DMARDs and thus had persistent disease, similar seroreversion rates were observed.18,25-27
Thus, seroreversion rates observed here are in line with previous findings in RA and are not increased in patients with sustained DMARD-free status.
When analyses were repeated in patients with high autoantibody levels (above the median) at baseline, seroreversion for anti-CCP2 IgG was not observed anymore, and seroreversion for RF IgM was less frequent than in the whole group of autoantibody-positive patients. This suggests that the observed seroreversion rates were mainly the result of patients who fluctuated around the cut-off level, and therefore that true seroreversion of ACPA and RF is only sporadically observed. Although no association between remission and seroreversion was observed, patients who achieved a DMARD-free status had a larger decrease in RF levels during follow-up than patients who did not achieve this outcome. The slopes of the levels along follow-up are relevant to appreciate the immunological evolution, which was different for RF and ACPA. Several studies have shown that improvement in disease activity is accompanied by decrease in RF levels,27-31 although some other
studies did not observe this.18,19,32 A unique feature of this study is that a prolonged
period of absence of clinical synovitis is observed, which is a much more stringent outcome than improvement in disease activity scores. Previous studies showed no relation between ACPA levels and disease activity and our data also demonstrated no relation with sustained DMARD-free status.27-31
Previously, it was shown that anti-CCP2 IgM remains present in RA patients during treatment in a persistent disease phase, suggesting that the anti-CCP immune response is continuously reactivated.33 Interestingly, we have shown
here that anti-CCP2 IgM also remained persistently present in patients who achieved sustained DMARD-free remission. This suggests that even in patients who are clinically cured, the anti-CCP response is persistently activated. ACPA characteristics other than level and IgM and IgG isotypes were not investigated. However, it is known that ACPA-level is highly associated with ACPA fine specificity and the number of ACPA isotypes.34 Based on this, it can be presumed that these
whereas for RA development it is not yet elucidated whether ACPA play a role in the pathophysiology or act as bystander, the present data suggest that the ACPA response does not explain the maintained resolution of clinical disease. Whether other immunological markers, for instance, changes in characteristics of autoantibody expressing B cells, associate with this phenotypic outcome, and therefore would be a better definition of immunological remission, is subject of further research.
A strength of this study is that patients had a long follow-up period also after DMARD cessation (median 4.2 years after achievement of sustained DMARD-free remission, thus median 5.2 years after DMARD stop). This follow-up time supports the validity of the outcome, as patients with an early flare after DMARD stop were never considered to be in remission, and also patients with a late flare were identified and excluded from the group of patients who achieved sustained DMARD-free remission. Late flares generally occurred 2.2 years after DMARD cessation and the large majority of patients were followed for a longer period of time after DMARD stop. A subanalysis in the patients who were followed for >4 years after achieving remission showed similar results, showing the robustness of the data in this respect. Of course, we do not know if the autoantibody-positive patients will get a flare of disease after an even longer follow-up period; this is subject of future studies. Some of the patients have been discharged from the outpatient clinic because of prolonged absence of synovitis and symptoms. Importantly, it is plausible that if symptoms will recur patients will return to our clinic since the Leiden University Medical Center is the only referral centre in a healthcare region of ∼400,000 inhabitants and has very easy access services, allowing that patients with symptoms suspicious of RA are seen within 1 week.35
Treatment was not protocolised and the applied treatments changed over time. This resulted in heterogeneity of treatments received by patients. Nonetheless, when stratifying patients in groups according to medications ever used during follow-up, results remained similar.
Another limitation might be that not of all patients with sustained DMARD-free remission serum samples were available after medication was stopped. However, when analyses were repeated in this subgroup of patients, similar results were obtained (data not shown).
Previously, it was suggested that remission can be defined according to different
conditions and presence of immunological remission, defined as the disappearance of autoantibodies, was suggested to be the deepest form of remission. In this long-term study, we were able to analyse a large number of ACPA-positive patients who achieved sustained DMARD-free remission. In this unique dataset, we observed that disappearance of autoantibodies rarely occurred, and that patients who achieved the best possible outcome of RA did not become more often seronegative than patients with persistent disease. Therefore, in our view, this definition of immunological remission should not be a long-term treatment target.
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Supplementary material
Supplementary table 9.1 Comparison of seroreversion rates between patients with sustained DMARD-free remission and with persistent RA, stratified for medication use during follow-up
DMARD-free sustained remission Flare after DMARD-free remission Persistent RA p-value (Fisher exact) Methotrexate+
ACPA+ n=90 anti-CCP2 IgG seroreversion, n(%) 6 (14) 1 (8) 2 (6) 0.64 Methotrexate+ RF+ n=116 RF IgM seroreversion, n(%) 10 (17) 1 (6) 6 (15) 0.71 Sulfasalazine+
ACPA+ n=41 anti-CCP2 IgG seroreversion, n(%) 3 (19) 0 (0) 1 (5) 0.48 Sulfasalazine+ RF+ n=48 RF IgM seroreversion, n(%) 3 (16) 0 (0) 4 (16) 1.00 Hydroxychloroquine+
ACPA+ n=43 anti-CCP2 IgG seroreversion, n(%) 2 (18) 0 (0) 1 (4) 0.31 Hydroxychloroquine+ RF+ n=58 RF IgM seroreversion, n(%) 6 (26) 0 (0) 1 (3) 0.05 TNF-inhibitor +
ACPA+ n=23 anti-CCP2 IgG seroreversion, n(%) 2 (25) 1 (100) 0 (0) 0.02 TNF-inhibitor +
RF+ n=26 RF IgM seroreversion, n(%) 0 (0) 0 (0) 2 (13) 0.63
Patients were stratified for medication use during follow-up. The duration of treatment and combination therapy is not indicated here. Numbers indicate the number of patients with CCP2 IgG or RF IgM seroreversion within patients who where ACPA-positive or RF-positive at disease presentation, and who used the indicated medication during follow-up. ACPA, citrullinated protein antibodies; CCP anti-cyclic citrullinated peptide 2; DMARD, disease-modifying antirheumatic drug; RA, rheumatoid arthritis; RF, rheumatoid factor; TNF, tumor necrosis factor.
Supplementary table 9.2 Comparison of seroreversion rates between patients with sustained DMARD-free remission and with persistent RA, stratified for baseline autoantibody status
Sustained DMARD-free remission No sustained DMARD-free remission p-value (Fisher exact) A ACPA+RF+
anti-CCP2 IgG reversion to negative, n(%) 4 (11) 2 (5) 0.41 anti-CCP2 stable positive, n(%) 34 (89) 41 (95)
RF IgM reversion to negative, n(%) 4 (11) 1 (2) 0.18 RF IgM stable positive, n(%) 34 (89) 42 (98)
ACPA+RF-anti-CCP2 IgG reversion to negative, n(%) 2 (22) 1 (25) 1.00 anti-CCP2 stable positive, n(%) 7 (78) 3 (75)
ACPA-RF+
RF IgM reversion to negative, n(%) 10 (30) 6 (43) 0.51 RF IgM stable positive, n(%) 23 (70) 8 (57)
B ACPA+RF+
anti-CCP2 IgG reversion to negative, n(%) 2 (7) 2 (6) 1.00 anti-CCP2 stable positive, n(%) 28 (93) 30 (94)
RF IgM reversion to negative, n(%) 11 (37) 12 (37.5) 1.00 RF IgM stable positive, n(%) 19 (63) 20 (62.5)
ACPA+RF-anti-CCP2 IgG reversion to negative, n(%) 4 (24) 1 (7) 0.34 anti-CCP2 stable positive, n(%) 13 (76) 14 (93)
ACPA-RF+
RF IgM reversion to negative, n(%) 4 (29) 3 (50) 0.61 RF IgM stable positive, n(%) 10 (71) 3 (50)
Patients were stratified for autoantibody status at baseline. In part A the cut-off for RF positivity was 8 IU/ml, in part B this was 33 IU/ml to arrive at a specificity of 98%, comparable to the ACPA test. Numbers indicate the number of patients with CCP2 IgG or RF IgM seroreversion within patients who where ACPA-positive or RF-ACPA-positive at disease presentation. ACPA, citrullinated protein antibodies; CCP anti-cyclic citrullinated peptide 2; DMARD, disease-modifying antirheumatic drug; RA, rheumatoid arthritis;
